The objective of this revised proposal is to investigate plumbagin, a medicinal plant-derived naphthoquinone, in the prevention of growth and metastasis of hormone refractory prostate cancer (PCa). Hormone refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. The roots of Plumbago zeylanica L. have been used as medicine in India for more than 2500 years in treatment of various ailments. However, no study exits on the effects of plumbagin in the prevention and/or treatment of PCa progression. Our observations that prompted the exploration of plumbagin in the prevention of growth and metastasis of PCa include: A) Plumbagin inhibits invasion of androgen-independent (AI) PCa cells. B) Plumbagin administration delays ectopic growth of hormone refractory PCa cells as well as reduces both tumor weight and volume by 90%. C) In AI PCa cells, plumbagin inhibits the constitutive activation of Stat3, NF-kB, AP-1 and expression of protein kinase C5 (PKC5), the predictive biomarkers of PCa aggressiveness. We now propose to test two hypotheses: 1) Plumbagin prevents the growth and metastasis of hormone refractory PCa. 2) The key mechanism of AI PCa prevention involves the ability of plumbagin to inhibit: i) expression levels as well as activation of PKC5 and PKC5 downstream protein kinase cascades, ii) AR expression and activation and iii) Stat3 activation as well as Stat3-regulated gene expression linked to AI PCa cell survival and metastasis. Two specific aims are proposed to test the hypotheses. Specific Aim #1: to determine whether plumbagin prevents the progression and metastasis of hormone refractory PCa. To accomplish this, we propose to determine the effects of plumbagin on: 1A) the growth and metastasis of orthotopic xenografts of Luciferase- labeled AI PCa cells (C4-2B) in SCID mice. Luciferase-labeled cells will be implanted into the prostate and the metastatic dissemination pattern of PCa to various tissues including bone will be determined by bioluminescence imaging of live mice and by real time polymerase chain reaction (RT-PCR) assay for luciferase gene using isolated tissue from sacrificed mice. B) The progression and metastasis of AI PCa in a PCa mouse model (bigenic Nkx3.1-/-//Pten-/+ mice). The bigenic Nkx3.1-/-//Pten-/+ mice recapitulate stages of human prostate cancer progression. Specific Aim #2: To determine whether the mechanism of PCa prevention by plumbagin involves its ability to inhibit: i) PKC5, ii) androgen receptor (AR) and iii) Stat3 activation as well as Stat3-regulated gene expression. To accomplish this, we propose to: 2A) obtain first correlative evidence, by analysis of specimens collected in experiments proposed under Specific Aim 1, whether plumbagin-caused inhibition of PCa associates with expression levels of activated PKC5, PKC5 downstream protein kinase cascade, AR and Stat3 activation as well as Stat3-reulated gene expression. 2B) determine precisely the link of PKC5 and its associated signals using AD PCa cell line LNCaP and its AI derivative C4-2B. Plumbagin will be a novel agent to prevent and treat AI PCa.